Use of betel leaf extract to induce IFN-gamma production from human peripheral blood T cells and as a Th1 type immunomodulator

ABSTRACT

This invention relates to use of betel leaf extract to induce IFNγ from human peripheral blood mononuclear cells and as a Th 1  type immuno modulator, wherein the said method comprises the steps of, preparing water extract of betel leaf, preparation of human peripheral blood mononuclear cells, incubation of hPBMC with betel leaf extract for a period of 18-48 hours, extraction of RNA for cytokine specific RT-PCR or for flow cytometry for the detection of intracellular cytokine protein, subjecting RNA for RT-PCR to obtain PCR products using IFNγ specific known primers, and enhancement of IFNγ as reflected by IFNγ specific band.

FIELD OF THE INVENTION

[0001] This invention relates to use of betel leaf extract to induceIFN-gamma production from human peripheral blood T cells.

BACKGROUND AND PRIOR ART REFERENCES

[0002] Stinging nettle leaf extracts are registered in Germany foradjuvant therapy of rheumatic diseases. In a whole blood culture system,the nettle extract IDS 23 (Rheuma-Hek) inhibited lipopolysaccharidestimulated monocyte cytokine expression, indicating an immunomodulatingeffect. (Antirheumatic Effect of IDS 23, a Stinging Nettle Leaf Extract,on in vitro expression of T helper cytokines). The applicantsinvestigated the immunomodulating effects of betel leaf extracts onphytohemagglutinin (PHA) stimulated peripheral blood mononuclear cells(PBMC) in vitro. Betel leaves has a strong pungent aromatic flavor andwidely used as a masticatory. Generally, mature or over mature leaves,which has ceased growing but not yet become brittle are used forchewing. The basic preparation for chewing purposes consists of betelleaf smeared with hydrated lime and catechu to which scrapings ofarecanut are added; flavorings such as coconut shavings, clove,cardamon, fennel, powdered liquorice, nutmeg and also tobacco are usedaccording to one's taste. In some places prepared pan is covered withsilver or gold leaf. As a masticatory, it is credited with manyproperties: it is aromatic, digestive, stimulant and carminative.Medicinally it is useful in catarrhal and pulmonary affections; it isalso used for poultices. The effects of chewing of betel with arecanutand other adjuncts are the excitation of the salivary glands and theirritation of the mucous membrane of the mouth. The red colorationproduced is due to a pigment in the arecanut, which manifests itselfunder the action of alkali in lime and catechu. A mild degree ofstimulation is produced, resulting in a sensation of warmth and wellbeing, besides imparting a pleasant odor. The most important factordetermining the aromatic value of the leaf is the amount andparticularly the nature of the essential oil present. Betel leaves fromdifferent regions vary in smell and taste. The most pungent is theSanchi type, while the most mild and sweet ones are from Madras. Thebetel leaves contain essential oils, the content of oil varies from 0.7to 2.6 per cent depending upon the varieties of leaves. The oil consistsof phenols and terpens. The higher the proportion of phenol oil, thebetter the quality. An isomer of eugenol named chavibetol (betel phenol;4-allyl-2-hydroxy-1-methoxy benzene) is considered to be thecharacteristic constituent of betel oil. It is however, absent in Indiansamples. Betel oil of Indian types contain as a predominant phenolicconstituent. Oil of betel has been used in the treatment of variousrespiratory catarrhs, under as a local application either by gargle orby inhalation in diphtheria. It has carminative properties. It exhibitsin different action on the central nervous system of mammals; lethaldoses produce deep narcosis leading to death within a few hours. Theessential oil and extracts of the leaves possess activity againstseveral Gram-positive and Gram-negative bacteria such as Micrococcuspyogenes var. albus and var. aureus, Bacillus subtilis and B.megaterium, Diplococcus pneumoniae, Streptococcus pyogenes, Escherichiacoli, Salmonella typhosa, Vibrio comma, Shigella dysenteriae, Proteusvulgaris, Pseudomonas solanacaerum, Sarcina lutea and Erwiniacarotovora. The oil is found to be lethal in about 5 minutes to theprotozoa Paramaecium caudatum (Wealth of India, Vol.8 pp.84-94). Itinhibits the growth of Vibrio cholerae, Salmonella typhosum and Shigellaflexneri and Escherichia coli. Steam—distillate of the leaves showedactivity against Mycobacterium tuberculosis.

OBJECTS OF THE INVENTION

[0003] The main object of the present invention is to provide a methodfor inducing IFN-γ from human peripheral blood mononuclear cells.

[0004] Another object of the present invention relates to use of betelleaf extract for inducing IFN-γ production from human peripheral blood Tcells.

[0005] Another object of the invention is to provide a compositioncomprising betel leaf extract, which is useful as Th₁ typeimmunomodulator.

SUMMARY OF THE INVENTION

[0006] To meet the above objects, the present invention provides amethod for inducing IFN-γ from human peripheral blood mononuclear cells.The invention also relates to use of betel leaf extract for inducingIFN-γ production from human peripheral blood T cells.

DETAILED DESCRIPTION OF THE INVENTION

[0007] Accordingly, the invention relates to a method for inducing IFNγfrom human peripheral blood mononuclear cells wherein the said methodcomprising preparing water extract of betel leaf; preparation of humanperipheral blood mononuclear cells; incubation of hPBMC with betel leafextract for a period of 18-48 hours; extraction of RNA for cytokinespecific RT-PCR or for flow cytometry for the detection of intracellularcytokine protein; subjecting RNA for RT-PCR to obtain PCR products usingIFNγ specific known primers and enhancement of IFNγ as reflected by IFNγspecific band.

[0008] Alternatively, one more method for inducing IFNγ produced fromhuman peripheral blood mononuclear cells wherein the said processcomprising subjecting incubated cells for intracellular staining forIFNγ; analysis of stained cells in flow cytometer; and enhancement ofIFNγ positive cells to at least seven fold.

[0009] A method for using betel leaf extract as Th₁ typeimmuno-modulator wherein the said method comprising:

[0010] a) administering to a subject at least 5 to 10 mg/ml/kg body wt.of betel leaf extract, and

[0011] b) administering the extract through oral or intramuscular routeonce in a day for a period of at least one month,

[0012] In an embodiment of the present invention provides apharmaceutical composition useful as Th₁ type immunomodulator, saidcomposition comprising an effective amount of betel leaf aqueous extractor lyophilized betel leaf extract together with or associated with anadditive.

[0013] In still another embodiment of the invention, the additive isselected in such a manner that does not interfere with the activity ofbetel leaf extract.

[0014] Yet another embodiment of the invention, the additive is selectedfrom nutrients such as proteins, carbohydrates, sugar, talc, magnesiumstearate, cellulose, calcium carbonate, starch-gelatin paste and/orpharmaceutically acceptable carriers, excipient, diluent or solvent.

[0015] Yet another embodiment of the invention, the aqueous extract,lyophilized product or the composition is administered orally orintramuscularly.

[0016] Yet another embodiment of the invention, the oral route is in theform of capsule, syrup, concentrate, powder or granules.

[0017] Yet another embodiment of the invention, the ratio of betel leafextract to the additive is in the range between 10-1:1-10

[0018] Yet another embodiment of the invention, the betel leaf extract,lyophilized extract or the composition comprising the betel leaf extractis administered at a dosage level between 5 to 10 mg/kg of body weightat least once in a day for one month.

[0019] In one more embodiment of the present invention, a method oftreating a subject to provide Th1 type immuno-mudulation, said methodcomprising administering a pharmaceutically effective amount of betelleaf extract, lyophilized extract or a composition comprising theextract to the subject.

[0020] Yet another embodiment of the present invention, the additive isselected in such a manner it does not interfere with the activity oflyophilized betel leaf extract.

[0021] Yet another embodiment of the invention, the additive is selectedfrom nutrients such as proteins, carbohydrates, sugar, talc, magnesiumstearate, cellulose, calcium carbonate, starch-gelatin paste and/orpharmaceutically acceptable carriers, excipient, diluent or solvent.

[0022] Yet another embodiment of the invention, the aqueous extract,lyophilized product or the composition is administered orally orintramuscularly.

[0023] Yet another embodiment of the invention, the oral route is in theform of capsule, syrup, concentrate, powder or granules.

[0024] Yet another embodiment of the invention, the ratio of betel leafextract to the additive is in the range between 10-1:1-10

[0025] Yet another embodiment of the invention, the betel leaf extract,lyophilized extract or the composition comprising the betel leaf extractis administered at a dosage level between 5 to 10 mg/kg of body weightat least once in a day for one month.

[0026] In one more embodiment of the present invention, use of betelleaf extract in association with suitable carriers/additive as Th₁ typeimmunomodulator.

[0027] Yet another embodiment of the present invention, the additive isselected in such a manner it does not interfere with the activity oflyophilized betel leaf extract.

[0028] Yet another embodiment of the invention, the additive is selectedfrom nutrients such as proteins, carbohydrates, sugar, talc, magnesiumstearate, cellulose, calcium carbonate, starch-gelatin paste and/orpharmaceutically acceptable carriers, excipient, diluent or solvent.

[0029] Yet another embodiment of the invention, the aqueous extract,lyophilized product or the composition is administered orally orintramuscularly.

[0030] Yet another embodiment of the invention, the oral route is in theform of capsule, syrup, concentrate, powder or granules.

[0031] Yet another embodiment of the invention, the ratio of betel leafextract to the additive is in the range between 10-1:1-10

[0032] Yet another embodiment of the invention, the betel leaf extract,lyophilized extract or the composition comprising the betel leaf extractis administered at a dosage level between 5 to 10 mg/kg of body weightat least once in a day for one month.

[0033] One more embodiment of the present invention relates to thepreparation of betel leaf extracts comprising the following steps;

[0034] 1) washing of the fresh leaves of Piper betel and homogenizing ina mixture blender;

[0035] 2) sonicating in an ultrasonic bath with 2 to 3 bursts each for15 minutes and filtering the extract, if desired repeating theextraction at least once and drying; and

[0036] 3) lyophilizing the extract to get a semi-solid mass

[0037] In one more embodiment of the invention, the lyophilized extractis obtained by freeze drying the aqueous extract by conventionalmethods.

[0038] In another embodiment, the water extract is prepared fromfollowing types of betel leaf (Piper betel) namely Wild type, Climbertype, Bangla type and Sweet type.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

[0039]FIG. 1: represents RT-PCR to demonstrate that betel leaf extractenhances IFN-γ mRNA expression by peripheral blood mononuclear cells(PBMC) of normal individuals.

[0040]FIG. 2: represents flow cytometric determination that betel leafextract enhances IFN-γ expression at the protein level by PBMC of normalindividuals.

[0041] The following examples further illustrate the invention, but theinvention is not limited thereto.

EXAMPLE 1

[0042] 34.14 gm of fresh leaves of Piper betle thoroughly washed insterile water was homogenized with 100 ml of glass distilled water in amixture-blender. It was then sonicated in an ultrasonic bath with 3burst each for 15 min. The extract was filtered through Whatman No.1filter paper and the filtrate was collected. This process of extractionwas repeated three times. The combined extract was lyophilized yieldinga semi-solid mass weighing 1.17 gm. This was then tested for biologicalactivity.

EXAMPLE 2

[0043] The fresh leaves of Piper betle weighing 21.68 gm homogenizedwith distilled water (60 ml) in a mixture-blender and then sonicated inan ultrasonic bath with 2 burst each for 15 min. It was allowed to beextracted overnight or 16 hours. Filtering through Whatman No.1 filterpaper separated the material extracted in water. This type of treatmentfor extraction was repeated for three times. The combined extract wasevaporated to dryness in a flash evaporator under reduced pressure at45° C. The residual substance was then dried in a desiccator under highvacuum and the semi-solid mass weighing 0.59 gm was tested forbiological activity.

Properties of the Materials

[0044] The biologically active material obtained by examples 1 and 2 hasthe following properties:

[0045] 1) The dried semisolid prepared as stated above was a darkcolored material soluble in water and dimethyl sulfoxide.

[0046] 2) Thin layer chromatography of the active material shows fivespots having R_(f)0.75, 0.64, 0.50, 0.40 and 0.33 in the solvent systemof n-butanol, acetic acid and water in the ratio of 9:5:7 respectively.

[0047] 3) The HPLC analysis of the active material using Intersil ODS-3(4.6×250 mm) analytical column, solvent system methanol and water in theratio of 4:1 and a flow rate of 1.0 ml/min., detection at 217 nmresolved the material into eleven peaks with the retention time of 2.69,4.27, 5.95, 6.97, 7.49, 9.39, 11.20, 12.40, 15.53, 18.90 and 21.49 mins.

EXAMPLE 3

[0048] 1. Preparation of Human Peripheral Blood Mononuclear Cells(PBMC):

[0049] Heparinized whole bloods (collected from normal individuals) weresubjected to Ficoll Hypaque density gradient centrifugation. Cells inthe interface were washed twice with phosphate buffered saline (PBS) andthen re-suspended in medium RPMI−1640 supplemented with 10% Fetal BovineSerum.

[0050] 2. Incubation of hPBMC with Betel Leaf Extract:

[0051] PBMC (5.0×10⁶ cells) were cultured overnight (18 hours) at 37° C.in 5% CO₂ in a total volume of 2.0 ml RPMI+10% FBS containing 5 μg/ml ofphytohemagglutinin (PHA) in 24 well plates in the presence or absence ofbetel leaf extracts (12.5 mg/ml final concentration). At the end of theincubation period PBMC were washed twice with PBS and used forextraction of total RNA for cytokine specific RT-PCR or for flowcytometry for the detection of intracellular cytokine at the single celllevel.

[0052] 3. RNA Preparation and RT-PCR

[0053] Total cellular RNA from cultured PBMC was extracted by Trizol(Gibco BRL). 5×10⁶ cells were cultured for 18 hours as described aboveand harvested and resuspended in 1 ml Trizol. 2 μg of RNA and 10 μm ofeach primers were used to synthesize cDNA and PCR in single tube usingsuperscript™ One step RT-PCR system (Gibco BRL) in a total volume of 25μl-Primer sequences of human IFNγ and human IL-4 were as follows:

[0054] IFNγ sense, 5′ TCT GCA TCG TTT TGG GTT CT 3′, antisense 5′ CAGCTT TTC GAAGTC ATC TC 3′; IL-4 Sense 5′ CCT CTG TTC TTC CTG CTAGC 3′;antisense 5′ CCG TTTCAGGAA TCG GAT CA 3′. Amplification and cDNAsynthesis was performed as described in manufacturer's protocol. PCRproducts were electrophoresed in a 1.2% agarose gel in the presence ofethidium bromide and photographed under ultraviolet transmilluminator.Molecular weight markers (123 bp ladder, Gibco BRL) were included in allgels. (J. Rheumatol. 1999: 26: 2517-2522)

[0055] 4. Flow Cytometry

[0056] Cells were washed, permeabilized by treatment with 4%paraformaldehyde for 10 min., followed by incubation with 0.1% saponinfor 10 min. Cells were then washed with washing buffer (PBS containing1% albumin, 0.1% saponin and 0.1% sodium azide). After washing,permeabilized cells were treated with FITC or PE labeled controlmonoclonal antibody (mAb), anti-human IFNγ Ab labeled with FITC oranti-human IL-4 mAb labeled with PE, for 20 min. in room temperature atdark. Cells were then washed once with washing buffer and once with PBSand then resuspended in PBS containing 1% paraformaldehyde for flowcytometric analysis.

[0057] Results:

[0058] Influence of Betel Leaf Extract on Th₁ Cytokine Expression

[0059] It is clear that FIG. 1, that betel leaf extract enhances IFNγmRNA expression but has no effect on IL-4 m RNA expression by peripharalblood mononuclear cells of normaL human individuals as determined byRT-PCR. In other words, the applicants' data as shown in FIG. Iindicates that betel leaf extract significantly enhances synthesis ofIFNγ specific mRNA and having virtually no effect on IL-4 mRNAsysnthesis.

[0060] IFNγ synthesis by PBMC was also detectable at the protein levelas evident from the flow cytometric dot plot analysis as shown in FIG.II. Only 0.9% PBMC were positive for IFNγ when PBMC were incubated withPHA (FIG. IIA). On the other hand, 7.1% PBMC showed intracellular IFNγ(FIG. IIB) when PBMC were incubated with PHA plus betel leaf extract. Incontrast, percentages of IL-4 producing cells were not appreciablychanged when PBMC were incubated with betel leaf extract (FIG. 2C & D).

[0061] Discussion

[0062] T cells are subdivided into Th₁ and Th₂ phenotypes by theircytokine patterns (Mossmann, T. R., Coffman, R L. Th₁ and Th₂ cells:different patterns of lymphokine secretions lead different functionalproperties. Annual Rev. Immunol. 1989, 7; 145-73), which regulate cellmediated and humoral immune responses. Inflammatory immune responses areprimarily mediated by Th₁ cell polpulations through the production ofIL₂ & IFN-γ, which enhance cellular immunity (Trinchieri G.Interlenkin-12 and its role in generation of Th₁ cells. Immunol Today1993; 14: 335-8; Germann, T., Szeliga, J., Hess, H. et al.Administration of interlenkin-12 in combination with type-II collageninduces severe arthritis in DBA/1 mice. Proc Natl Acad Sci. USA 1995;92: 4823-7).

[0063] Thus, our experimental results suggest that betel leaf extractsenhance Th₁ type response leading to enhanced cellular immunity.

1 4 1 20 DNA Artificial Sequence IFNgamma-sense 1 tctgcatcgt tttgggttct20 2 20 DNA Artificial Sequence IFNgamma-antisense 2 cagcttttcgaagtcatctc 20 3 20 DNA Artificial Sequence IL4-sense 3 cctctgttcttcctgctagc 20 4 20 DNA Artificial Sequence IL4-antisense 4 ccgtttcaggaatcggatca 20

1. A method for inducing IFNγ from human peripheral blood mononuclearcells wherein the said method comprises the steps of: a) preparing waterextract of betel leaf, b) preparation of human peripheral bloodmononuclear cells, c) incubation of hPBMC with betel leaf extract for aperiod of 18-48 hours, d) extraction of RNA for cytokine specific RT-PCRor for flow cytometry for the detection of intracellular cytokineprotein, e) subjecting RNA for RT-PCR to obtain PCR products using IFNγspecific known primers, and f) enhancement of IFNγ as reflected by IFNγspecific band.
 2. A method for inducing IFNγ produced from humanperipheral blood mononuclear cells wherein the said process furthercomprises alternate steps of: a) subjecting incubated cells forintracellular staining for IFNγ, b) analysis of stained cells in flowcytometer, and c) enhancement of IFNγ positive cells to at least sevenfold.
 3. A method of treating a subject to provide Th1 typeimmuno-modulation, said method comprising administering apharmaceutically effective amount of betel leaf extract, lyophilizedextract or a composition comprising the extract to the subject.
 4. Amethod as claimed in claim 3 wherein, the composition comprising betelleaf extract associated with or in combination with a pharmaceuticallyacceptable additive.
 5. A method as claimed in claim 3 wherein, theadditive is selected in such a manner it does not interfere with theactivity of lyophilized betel leaf extract.
 6. A method as claimed inclaim 3 wherein, the additive is selected from nutrients such asproteins, carbohydrates, sugar, talc, magnesium stearate, cellulose,calcium carbonate, starch-gelatin paste and/or pharmaceuticallyacceptable carriers, excipient, diluent or solvent.
 7. A method asclaimed in claim 3 wherein, the aqueous extract, lyophilized product orthe composition is administered orally or intramuscularly.
 8. A methodas claimed in claim 3 wherein, the oral route is in the form of capsule,syrup, concentrate, powder or granules.
 9. A method as claimed in claim3 wherein, the ratio of betel leaf extract to the additive is in therange between 10-1:1-10
 10. A method as claimed in claim 3 wherein, thebetel leaf extract, lyophilized extract or the composition comprisingthe betel leaf extract is administered at a dosage level between 5 to 10mg/kg of body weight at least once in a day for one month.
 11. Apharmaceutical composition useful as Th₁ type immunomodulator, saidcomposition comprising an effective amount of betel leaf aqueous extractor lyophilized betel leaf extract together with or associated with anadditive.
 12. A composition as claimed in claim 11 wherein, thelyophilized extract is obtained by freeze drying the aqueous extract byconventional methods.
 13. A composition as claimed in claim 11 wherein,the additive is selected in such a manner that does not interfere withthe activity of betel leaf extract.
 14. A composition as claimed inclaim 11 wherein, the additive is selected from nutrients such asproteins, carbohydrates, sugar, talc, magnesium stearate, cellulose,calcium carbonate, starch-gelatin paste and/or pharmaceuticallyacceptable carriers, excipient, diluent or solvent.
 15. A composition asclaimed in claim 11 wherein, the aqueous extract, lyophilized product orthe composition is administered orally or intramuscularly.
 16. Acomposition as claimed in claim 11 wherein, the oral route is in theform of capsule, syrup, concentrate, powder or granules.
 17. Acomposition as claimed in claim 11 wherein the ratio of betel leafextract to the additive is in the range between 10-1:1-10
 18. Acomposition as claimed in claim 11 wherein, the betel leaf extract,lyophilized extract or the composition comprising the betel leaf extractis administered at a dosage level between 5 to 10 mg/kg of body weightat least once in a day for one month.
 19. A composition as claimed inclaim 11 wherein, the lyophilized betel leaf extract has the followingproperties: i) the dried sample is a dark colored material soluble inwater and dimethyl sulfoxide, ii) thin layer chromatography of theactive material shows five spots having R_(f)0.75, 0.64, 0.50, 0.40 and0.33 in the solvent system of n-butanol, acetic acid and water in theratio of 9:5:7 respectively, and iii) the HPLC analysis of the activematerial using Intersil ODS-3 (4.6×250 mm) analytical column, solventsystem methanol and water in the ratio of 4:1 and a flow rate of 1.0ml/min., detection at 217 nm resolved the material into eleven peakswith the retention time of 2.69, 4.27, 5.95, 6.97, 7.49, 9.39, 11.20,12.40, 15.53, 18.90 and 21.49 min
 20. A method for using betel leafextract as Th₁ type immuno-modulator wherein the said method comprising:a) administering to a subject at least 5 to 10 mg/ml/kg body wt. ofbetel leaf extract juice, b) administering extract at least once in aday for a period of at least one month, c) administering the extractthrough oral or intramusculor route.
 21. A use of betel leaf extract orits lyophilized extract or a composition comprising effective amount ofbetel leaf extract for inducing IFNγ from human peripheral bloodmononuclear cells or as a Th₁ type immunomodulator.
 22. Use as claimedin claim 21 wherein, the said composition comprising betel leaf extractassociated with or in combination with a pharmaceutically acceptableadditive.
 23. Use as claimed in claim 21 wherein, the additive isselected in such a manner it does not interfere with the activity oflyophilized betel leaf extract.
 24. Use as claimed in claim 21 wherein,the additive is selected from nutrients such as proteins, carbohydrates,sugar, talc, magnesium stearate, cellulose, calcium carbonate,starch-gelatin paste and/or pharmaceutically acceptable carriers,excipient, diluent or solvent.
 25. Use as claimed in claim 21 wherein,the aqueous extract, lyophilized product or the composition isadministered orally or intramuscularly.
 26. Use as claimed in claim 21wherein, the oral route is in the form of capsule, syrup, concentrate,powder or granules.
 27. Use as claimed in claim 21 wherein, the ratio ofbetel leaf extract to the additive is in the range between 10-1:1-10 28.Use as claimed in claim 21 wherein, the betel leaf extract, lyophilizedextract or the composition comprising the betel leaf extract isadministered at a dosage level between 5 to 10 mg/kg of body weight atleast once in a day for one month.